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617 Efficient Gene Delivery to the Enteric Nervous System by IV Delivery of AAV Vectors
617 Efficient Gene Delivery to the Enteric Nervous System by IV Delivery of AAV Vectors Sara E. Gombash Lampe, Christopher Cowley, Julie Fitzgerald, Christian Mueller, Fievos L. Christofi, Kevin Foust Gene therapy may be useful to treat and study the enteric nervous system (ENS). In this study, we characterized transduction efficiency and distribution in the myenteric and submucosal plexuses following intravenous (IV) injection of adeno-associated viral vectors (AAV) expressing GFP in mice. Neonate (P1) mice were injected with AAV9-CB-GFP (3 x 1011 vg) in the temporal vein and juvenile mice (P21) received tail vein injections (2 x 1012 vg). Mice were euthanized at 60-90 days of age, and the myenteric plexus was examined. GFP positive myenteric neurons were found along the entire GI tract in injected mice. Segmental examinations of myenteric neurons in the colon showed no oral-aboral bias. Neuronal transduction ranged from 21-57% depending on the region. GFP co-localized with interneurons, excitatory and sensory myenteric neurons. In contrast, GFP expression in VIP, nNOS and glial cells was absent. However, IV injection of AAV9 with a glial promoter (GFAP) drove GFP expression exclusively in glia. Neonate IV injections with AAV1, 5, and 6 containing the CB-GFP expression cassette produced little to no transgene expression in the myenteric plexus. However, AAV8-CB-GFP showed significantly higher neuronal transduction compared to AAV9 injected mice. AAV8-CB-GFP expressed in all ENS cell types examined including VIP and nNOS neurons and intraganglionic S100 glia. Characterization of AAV-CB-GFP transduction in submucosal neurons is ongoing. These data demonstrate that the ENS is efficiently targeted by AAV8 and AAV9 following IV delivery. Further, transgene expression can be customized with changes in the viral capsid and expression cassette.
619 Single-Electrode Colon Stimulation and Impedance Monitoring in an Intestinal Aganglionosis Model Yi-Kai Lo, Justin Wagner, Chih-Wei Chang, Joshua D. Rouch, James Dunn, Wentai Liu Background: Enteric neuromuscular dysfunction is the defining characteristic in the pathophysiology of Hirschsprung's disease, esophageal achalasia, Chagas disease, and gastroparesis. Medical therapies for these disorders are limited while surgical treatment may incur significant morbidity with variable outcomes. Electrical stimulation (ES) of intestine has been described with inconclusive clinical benefit. In this experimental series, we propose a novel strategy to stimulate aganglionic intestine while simultaneously monitoring myenteric impedance using the same electrode. Tissue impedance variation (TIV) provides a quantifiable feedback when ES is applied to intestinal tissue. Method: This experiment was performed in 12 adult female Lewis rats: 4 underwent jejunal segment isolation and benzalkonium chloride (BAC) aganglionosis induction, 4 underwent colonic segment isolation and BAC aganglionosis induction, and 4 underwent sham laparotomy. A customized versatile multichannel stimulator was used to bi-phasically current stimulate normal and aganglionic intestinal segments in the same rat. TIV of induced segmental contraction and subsequent relaxation were recorded through same electrodes. Stimulation strategies included a long pulse width (PW), high-intensity stimulus to generate smooth muscle (SM) contraction and a short-PW, lowintensity stimulus for impedance measurement. TIV was measured at several stimulus settings to determine the effective parameters to induce SM contraction among aganglionic and normal segments. Results: Extrinsic stimuli generated peristaltic waves of contraction in normal jejunum and colon; only local contraction at the stimulus sites in aganglionic jejunum and colon. At 10 kHz, tissue impedance varied from 3.7-6.25 k V, correlating with the degree of contraction observed grossly. Sequential contractions of aganglionic SM were generated by electrically stimulating and recording impedance at multiple sites longitudinally along the intestinal segment. The chronological multi-site impedance change represented the propagation of peristaltic wave of SM contraction. In all 8 aganglionic jejunal and colonic segements, cathodic-first biphasic stimuli, with 1 ms PW, 1 mA current intensity, and 100 Hz frequency were able to generate SM contraction. The speed of the peristaltic wave can be measured accordingly using impedance method. Conclusion: We report efficient stimulation parameters to generate intestinal SM contraction with a PW significantly shorter than previously reported. Our results demonstrate impedance measurement is a non-invasive effective technique to monitor SM contraction. This method permits further study of gastrointestinal tract electrophysiology in normal and diseased segments, and enables the development of implanted stimulators to treat disorders of enteric neuromuscular dysfunction.
AAV9 mediated GFP expression in green. HuD, a myenteric neuron marker, is in red.
618 Effect of Anesthesia in Colon Motility: Prospective Study of Children With Intractable Constipation Undergoing Colon Manometry Ricardo A. Arbizu, Nicole Heinz, Maureen Amicangelo, Samuel Nurko, Leonel Rodriguez Introduction: Constipation is amongst the most common complaints to pediatricians and pediatric gastroenterologists. Colon manometry (CM) is a useful test to evaluate the pathophysiology of defecation disorders. Currently there is no standardization on its performance with some centers doing the study after anesthesia and others 24 hours later. Information on the effect of anesthesia in colon motility is limited, and its effect on CM interpretation is controversial. Methods: Prospective study evaluating the effect of anesthesia on colon motility and CM interpretation in children with intractable constipation. All patients received a bowel preparation the day before the study and underwent anesthesia with propofol (no narcotics) for catheter placement (6 also received rocuronium). Study started at least 1 hour after anesthesia with patient fully awake. CM was performed on the day of the catheter placement and repeated the next day. Study included an hour of fasting followed by meal and bisacodyl challenges and was interpreted as normal when gastrocolonic (GC) response to a meal and fully propagated high amplitude propagating contractions (HAPCs) were observed. To evaluate the effect of anesthesia on colon motility we compared the motility index of both days on each study phase (fasting, post-prandial and bisacodyl challenge) for left, right and whole colon. To evaluate the effect of anesthesia on CM interpretation we compared the GC response to a meal, quality and quantity of HAPCs and proportions of studies interpreted as normal between both days. Results: A total of 32 children were included, median age was 10.5 years (range 1.5-18 years) and 16 were female. We observed a significant effect of anesthesia in all the colon motility parameters (Table 1). We also found a higher number of HAPCs on day 2 vs. day 1 (10 vs. 6.5, p<0.001) and although we found no significant difference in the presence of HAPCs (p=0.15) between both days, we observed HAPCs on day 2 in 4/5 of the patients with absent HAPCs on day 1 and 3 of those 4 patients demonstrating fully propagated HAPCs on day 2. We found a higher proportion of normal HAPCs on day 2 vs. day 1 (25/32 or 78% vs. 17/32 or 53%, p=0.002). A total of 8/32 (25%) patients with an abnormal study on day 1 were interpreted as normal on day 2. We found no effect of rocuronium on colon motility and study interpretation but
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we found a higher median anesthesia time among those with no HAPCs on day 1 compared to those with HAPCs. All patients with normal study on day 1 remained normal on day 2. Conclusions: Anesthesia has a significant effect in colon motility leading to an incorrect interpretation of the CM as abnormal in up to 25% of patients. CM should be repeated the next day on those patients with an abnormal study on the day of the catheter placement under anesthesia. Table 1. Effect of Anesthesia on Colon Motility
619 Single-Electrode Colon Stimulation and Impedance Monitoring in an Intestinal Aganglionosis Model Yi-Kai Lo, Justin Wagner, Chih-Wei Chang, Joshua D. Rouch, James Dunn, Wentai Liu Background: Enteric neuromuscular dysfunction is the defining characteristic in the pathophysiology of Hirschsprung's disease, esophageal achalasia, Chagas disease, and gastroparesis. Medical therapies for these disorders are limited while surgical treatment may incur significant morbidity with variable outcomes. Electrical stimulation (ES) of intestine has been described with inconclusive clinical benefit. In this experimental series, we propose a novel strategy to stimulate aganglionic intestine while simultaneously monitoring myenteric impedance using the same electrode. Tissue impedance variation (TIV) provides a quantifiable feedback when ES is applied to intestinal tissue. Method: This experiment was performed in 12 adult female Lewis rats: 4 underwent jejunal segment isolation and benzalkonium chloride (BAC) aganglionosis induction, 4 underwent colonic segment isolation and BAC aganglionosis induction, and 4 underwent sham laparotomy. A customized versatile multichannel stimulator was used to bi-phasically current stimulate normal and aganglionic intestinal segments in the same rat. TIV of induced segmental contraction and subsequent relaxation were recorded through same electrodes. Stimulation strategies included a long pulse width (PW), high-intensity stimulus to generate smooth muscle (SM) contraction and a short-PW, lowintensity stimulus for impedance measurement. TIV was measured at several stimulus settings to determine the effective parameters to induce SM contraction among aganglionic and normal segments. Results: Extrinsic stimuli generated peristaltic waves of contraction in normal jejunum and colon; only local contraction at the stimulus sites in aganglionic jejunum and colon. At 10 kHz, tissue impedance varied from 3.7-6.25 k V, correlating with the degree of contraction observed grossly. Sequential contractions of aganglionic SM were generated by electrically stimulating and recording impedance at multiple sites longitudinally along the intestinal segment. The chronological multi-site impedance change represented the propagation of peristaltic wave of SM contraction. In all 8 aganglionic jejunal and colonic segements, cathodic-first biphasic stimuli, with 1 ms PW, 1 mA current intensity, and 100 Hz frequency were able to generate SM contraction. The speed of the peristaltic wave can be measured accordingly using impedance method. Conclusion: We report efficient stimulation parameters to generate intestinal SM contraction with a PW significantly shorter than previously reported. Our results demonstrate impedance measurement is a non-invasive effective technique to monitor SM contraction. This method permits further study of gastrointestinal tract electrophysiology in normal and diseased segments, and enables the development of implanted stimulators to treat disorders of enteric neuromuscular dysfunction.
AAV9 mediated GFP expression in green. HuD, a myenteric neuron marker, is in red.
618 Effect of Anesthesia in Colon Motility: Prospective Study of Children With Intractable Constipation Undergoing Colon Manometry Ricardo A. Arbizu, Nicole Heinz, Maureen Amicangelo, Samuel Nurko, Leonel Rodriguez Introduction: Constipation is amongst the most common complaints to pediatricians and pediatric gastroenterologists. Colon manometry (CM) is a useful test to evaluate the pathophysiology of defecation disorders. Currently there is no standardization on its performance with some centers doing the study after anesthesia and others 24 hours later. Information on the effect of anesthesia in colon motility is limited, and its effect on CM interpretation is controversial. Methods: Prospective study evaluating the effect of anesthesia on colon motility and CM interpretation in children with intractable constipation. All patients received a bowel preparation the day before the study and underwent anesthesia with propofol (no narcotics) for catheter placement (6 also received rocuronium). Study started at least 1 hour after anesthesia with patient fully awake. CM was performed on the day of the catheter placement and repeated the next day. Study included an hour of fasting followed by meal and bisacodyl challenges and was interpreted as normal when gastrocolonic (GC) response to a meal and fully propagated high amplitude propagating contractions (HAPCs) were observed. To evaluate the effect of anesthesia on colon motility we compared the motility index of both days on each study phase (fasting, post-prandial and bisacodyl challenge) for left, right and whole colon. To evaluate the effect of anesthesia on CM interpretation we compared the GC response to a meal, quality and quantity of HAPCs and proportions of studies interpreted as normal between both days. Results: A total of 32 children were included, median age was 10.5 years (range 1.5-18 years) and 16 were female. We observed a significant effect of anesthesia in all the colon motility parameters (Table 1). We also found a higher number of HAPCs on day 2 vs. day 1 (10 vs. 6.5, p<0.001) and although we found no significant difference in the presence of HAPCs (p=0.15) between both days, we observed HAPCs on day 2 in 4/5 of the patients with absent HAPCs on day 1 and 3 of those 4 patients demonstrating fully propagated HAPCs on day 2. We found a higher proportion of normal HAPCs on day 2 vs. day 1 (25/32 or 78% vs. 17/32 or 53%, p=0.002). A total of 8/32 (25%) patients with an abnormal study on day 1 were interpreted as normal on day 2. We found no effect of rocuronium on colon motility and study interpretation but
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we found a higher median anesthesia time among those with no HAPCs on day 1 compared to those with HAPCs. All patients with normal study on day 1 remained normal on day 2. Conclusions: Anesthesia has a significant effect in colon motility leading to an incorrect interpretation of the CM as abnormal in up to 25% of patients. CM should be repeated the next day on those patients with an abnormal study on the day of the catheter placement under anesthesia. Table 1. Effect of Anesthesia on Colon Motility